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ISSN: 0974-276X
Journal of
Ulmer et al., J Proteomics Bioinform 2015, 8:6 http://dx.doi.org/10.4172/jpb.1000360
Proteomics & Bioinformatics
Research Article Research Article
Open OpenAccess Access
Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells Candice Z Ulmer1, Richard A Yost1,2, Jing Chen2, Clayton E Mathews2 and Timothy J Garrett2* 1 2
Department of Chemistry, University of Florida, Gainesville, FL, USA Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA
Abstract Metabolomics is the comprehensive study of metabolism as it pertains to an organism or biological system. Lipidomics, a subset discipline of metabolomics, encompasses the study of cellular lipid functions: including pathways, networks, and interactions. The abundance of metabolites and lipids, along with their contribution to health and disease, makes metabolomics a valuable tool for biomarker research. Disease biomarker identification requires a reproducible, sensitive, and accurate analytical platform. Although transcriptomic and proteomic areas have well-established protocols for sample preparation and data processing, the metabolomics field is still developing comparable standardized conventions. Furthermore, of the few comparative LC-MS metabolomic studies that have been applied to mammalian cell cultures, most are targeted to adherent cell lines. The purpose of this work was to optimize a sample preparation workflow for the cellular metabolomic analysis of suspensioncultured mammalian cells using commercially available Jurkat T lymphocytes as a model system. The current investigation evaluated commonly used sample preparation techniques for reproducibility, accuracy, and applicability to untargeted biomarker discovery. Results show ammoniated cell rinsing solutions to be an effective means to remove extracellular components present in the media without causing ion suppression or affecting the integrity of the cellular membrane. Additionally, a novel workflow was designed to allow for the combined analysis of metabolites and lipids from mammalian suspension cells from a single cell pellet. The Folch lipid extraction protocol was coupled to an 80% MeOH metabolite isolation to ensure high extraction efficiency for phospholipids and triacylglycerides. While the workflow was tailored to cells in suspension, it could also be applied to adherent cell lines.
Keywords:
Sample preparation; Mammalian suspension cells
Metabolomics;
Lipidomics;
Abbreviations:
LC-MS: Liquid Chromatography-Mass Spectrometry; MeOH: Methanol; MTBE: Methyl Tert-Butyl Ether; ISTD: Internal Standard; CHCl3: Chloroform; H2O: Water; IPA: Isopropanol; TG: Triacylglyceride; LPC: Lysophosphatidylcholine; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PS: Phosphatidylserine; PG: Phosphatidylglycerol; BD: Bligh-Dyer; ABD: Acidified Bligh-Dyer
Introduction Metabolomics is the comprehensive study of metabolism as it pertains to an organism or living biological system in response to a stimulus, a pathophysiological condition, or genetic modification [15]. Lipidomics, is a subset discipline that encompasses the study of cellular lipid pathways, networks, functions, and interactions. Cellular metabolomics has evolved into an important technique for analyzing the biochemical response of metabolites and lipids within representative cell cultures [6]. Metabolites and lipids, although chemically and physically diverse (e.g., molecular weight, polarity, acidity, stability, and function), represent the naturally occurring biological events within a cell and the phenotypic state of a biological system [1-3,5,7-9]. Metabolism, regardless of the sample type, is an essential component of cellular function as it encompasses the up-stream interactions between the genome, transcriptome, and proteome [4,5,10]. Examination of involved endogenous metabolites and lipids provides insight into the affected metabolic pathways in biological processes and the fine detailed information of disease perturbations [2,4-6,10]. Mass spectrometry has developed into an analytical tool of choice in metabolomics due to increasing instrument speeds, wider dynamic J Proteomics Bioinform ISSN: 0974-276X JPB, an open access journal
signal ranges, and enhanced quantitative capabilities [11]. Coupling mass spectrometry with chromatographic separation methods such as liquid chromatography has allowed for reliable identification of lowlevel metabolites and lipids in complex mixtures [1,3,11,12]. Metabolomic approaches for biomarker discovery can include targeted or untargeted studies. The latter focuses on a comprehensive or global analysis of primary and secondary metabolites and/or lipids, whereas targeted studies analyze predefined selections of compounds originating from a specific metabolic pathway to better understand a biological event [3,9,12]. Sample preparation is the first step in ensuring reproducibility and accuracy for untargeted biomarker discovery. All samples undergoing mass spectrometric analysis should be prepared in an unbiased and reproducible manner that maintains the integrity of intracellular compounds [7]. Each ‘omics’ field (e.g., genomics, proteomics, metabolomics, and lipidomics) has its own unique considerations in sample preparation [13]. Because metabolomics is a relatively new discipline, standard protocols for sample preparation
*Corresponding author: Timothy J. Garrett, Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA, Tel: 1-352-273-5050; Fax: 352-294-4454; E-mail:
[email protected] Received April 24, 2015; Accepted Jun 01, 2015; Published June 05, 2015 Citation: Ulmer CZ, Yost RA, Chen J, Mathews CE, Garrett TJ (2015) Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells. J Proteomics Bioinform 8: 126-132. doi:10.4172/jpb.1000360 Copyright: © 2015 Ulmer CZ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Citation: Ulmer CZ, Yost RA, Chen J, Mathews CE, Garrett TJ (2015) Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells. J Proteomics Bioinform 8: 126-132. doi:10.4172/jpb.1000360
of suspension-cultured mammalian cells such as those available for proteomics and genomics analyses have not been fully vetted [2,5,13]. Additionally, cellular metabolomics protocols should be tailored for specific mammalian cell lines (adherent or suspension cells) [1,2,7,14]. Lipids must be extracted to investigate changes in composition influenced by cell growth, differentiation, and intracellular signaling processes [8]. Historically, the Folch lipid extraction method (2:1 chloroform/methanol mixture) was established to isolate brain lipids. This method along with the Bligh-Dyer lipid extraction method (1:2 chloroform/methanol mixture) are two of the most common extraction systems to recover lipids in the chloroform layer [8]. Both methods can be applied to cells. Previous research has shown other extraction methods such as a single-phase butanol, isopropanol/hexane mixture, or isopropanol/chloroform mixture to exhibit high extraction efficiency for cholesterol, but a low recovery for triacylglycerides. The chloroform/methanol mixture systems, compared to those previously listed, have high extraction efficiencies for phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and triacylglyceride (TG) groups [8]. Without comparative studies, it is difficult to evaluate sample-dependent procedures found in the literature for lipid recovery of a wide range of compounds [6]. Thus, this work presents a comparative evaluation of select mammalian cell sample preparation methods and lipid extraction procedures using relative LC-MS quantification of exogenous standards and endogenous compounds. These findings are used to develop an efficient and reproducible sample preparation workflow to measure cellular metabolite and lipid concentrations in an unbiased manner [15]. Because the metabolite and lipid contents are often analyzed from separate aliquots of a cell suspension, a novel workflow was designed to allow for the combined analysis of metabolites and lipids from a single cell pellet.
Materials and Methods Materials Roswell Park Memorial Institute (RPMI) 1640 medium (10-043CV), fetal bovine serum (FBS), phosphate buffered saline (PBS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1M) were purchased from Corning, Inc. Sodium pyruvate (100 mM), ammonium formate, sodium chloride, exogenous triacylglyceride (TG 15:0/15:0/15:0 and TG 17:0/17:0/17:0) lipid standards, and D-glucose were purchased from Sigma-Aldrich. Exogenous lysophosphatidylcholine (LPC 17:0 and LPC 19:0), phosphatidylcholine (PC 17:0/17:0 and PC 19:0/19:0), phosphatidylethanolamine (PE 15:0/15:0 and PE 17:0/17:0), phosphatidylserine (PS 14:0/14:0 and PS 17:0/17:0), and phosphatidylglycerol (PG 14:0/14:0 and PG 17:0/17:0) lipid standards were purchased from Avanti Polar Lipids. All lipid standards were diluted prior to analysis in 1:2 (v/v) chloroform:methanol (CHCl3:MeOH) and a working 100 ppm standard mix was then prepared by diluting the stock solution with the same solvent mixture. The following isotopic standards were purchased from C/D/N isotopes: creatine-D3 H2O (methyl-d3), D-leucine-D10, L-tryptophan-2,3,3-D3, and caffeine-D3 (1-methyl-d3). Ammonium acetate and all analytical grade solvents (formic acid, chloroform, methyl-tert butyl ether, and methanol) were purchased from Fisher-Scientific. All mobile phase solvents were Fisher Optima LC/MS grade (acetonitrile, isopropanol, and water).
Cell culture Jurkat cells (Clone E6-1) were purchased as a frozen cell culture J Proteomics Bioinform ISSN: 0974-276X JPB, an open access journal
from ATCC (Manassas, VA) and grown to confluence according to the manufacturer’s recommendations. Jurkat cells were maintained as a continuous cell culture (1 × 105 to 1 × 106 viable cells/mL) in sterile RPMI 1640 medium supplemented with 10% FBS, 5 mM D-glucose, 10 mM HEPES, and 1 mM sodium pyruvate. Cells were maintained at 37°C in an atmosphere containing 5% CO2. Cell viability was determined by the trypan blue exclusion test before reaching confluency and prior to metabolite/lipid extraction. Once confluent but still in the logarithmic growth phase, triplicate cell pellets were washed 3x with one of the ice cold rinsing solvents (0.3% ammonium acetate, 0.3% ammonium formate, 0.9% NaCl, 1 M PBS, and 100 mM HEPES), and centrifuged at 311 x g for 5 min. The supernatant was discarded and the cell pellet was dried under nitrogen. The dried cell pellet was stored at -80°C until extraction. Samples were kept on ice from collection to metabolite extraction to prevent metabolite turnover or a change in the metabolite profile.
Protein precipitation for metabolite isolation A 15 ppm exogenous, isotopically labeled metabolite standard mix (creatine-D3; D-leucine-D10; L-tryptophan-2,3,3-D3; and caffeine-D3) was spiked into cell pellets (9 μL) washed with one of the four rinsing solutions. The extraction buffer (ice cold 80% methanol, 2 mL) was added to the cell pellet. The suspension was incubated on ice for 5 min and centrifuged at 311 x g for 5 min. The supernatant was removed using a pipette, transferred to a new eppendorf tube, and then dried under nitrogen. Samples were reconstituted in 30 µL of 0.1% formic acid in water, vortexed briefly, and centrifuged at 311 x g for 5 min at 4°C to remove any insoluble components. The supernatant was transferred to an LC vial with a glass insert (Fisher Scientific) and then used for metabolomic analysis.
Lipid extraction from Jurkat T-lymphocyte cells Pelleted Jurkat cells were suspended in 0.3% ammonium formate. Twelve aliquots, each containing 1.5x107 cells in 600 μL were placed into 15 mL conical tubes. Three aliquots for each of the four solvent extraction volumes were extracted according to either the Folch [16], Bligh & Dyer [17], acidified Bligh & Dyer methods, or Matyash [7,18]. An exogenous lipid standard mix (15 ppm) containing 12 lipids was spiked (15 μL) into each aliquot pre-extraction. The relative abundance of the lipid standards was quantified by comparing the integrated peak area of each compound. [Folch method]: Chloroform:methanol (2:1, v/v) was used according to the original Folch procedure allowing for a CHCl3:MeOH:H2O ratio of 8:4:3 (v/v). Briefly, ice-cold methanol (0.5 mL) and chloroform (1 mL) were added directly to the cell pellet. The suspension was vortexed occasionally to bring about physical mixing and incubated on ice for 20-40 min. After the addition of water (0.38 mL), used to separate the aqueous and organic layer, the suspension was incubated on ice for an additional 10 min. Samples were centrifuged at 311 x g for 5 min at 4°C. The lower phase (organic) layer was transferred to a new tube. The aqueous layer was re-extracted with 2:1 chloroform/methanol. The chloroform layers were combined for analysis. Samples were then dried under nitrogen and reconstituted in 50 µL of isopropanol (IPA) and transferred to an LC vial with glass insert. [Bligh-Dyer (BD)]: Chloroform/methanol (1:2, v/v) was used according to the original procedure. The cell suspension was extracted by the addition of the chloroform/methanol mixture (1.5 mL of a 1:2, v/v), vortexed, and incubated on ice for 20 min. The mixture was washed with water (0.47 mL), vortexed, and centrifuged. The organic layer was collected and the aqueous layer was re-extracted with 1:1
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Citation: Ulmer CZ, Yost RA, Chen J, Mathews CE, Garrett TJ (2015) Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells. J Proteomics Bioinform 8: 126-132. doi:10.4172/jpb.1000360
chloroform/methanol (2 mL). The chloroform layers were combined for analysis. Samples were then dried under nitrogen and reconstituted in 50 µL of IPA.
lipid standards spiked into the reconstitution solvent was injected over the course of a batch (7-9 samples) to evaluate the mass accuracy (less than 0.8 ppm error) and instrument variability across the run.
[Acidified Bligh-Dyer (ABD)]: The cell suspension was extracted using the same procedure as the Bligh-Dyer, but with 25 µL of 3 M HCl added after the chloroform addition.
Data processing and statistical analysis
[Matyash method]: The cell suspension was extracted according to the procedure found in literature [18]. To each cell pellet, methanol (0.5 mL) and MTBE (1.7 mL) were added. The cell suspension was incubated at room temperature on an orbital shaker for 1 hr. After the addition of water to account for a final ratio of MTBE:MeOH:H2O (10:3:2.5, v/v/v), the mixture was incubated at room temperature again for 10 min, and centrifuged at 311 x g for 10 min. The organic (upper) layer was collected and the aqueous layer was re-extracted with 10:3:2.5 (v/v/v) MTBE:MeOH:H2O. The MTBE layers were combined for analysis. Samples were then dried under nitrogen and reconstituted in 50 µL of IPA.
All LC-MS data were collected and initially processed by the Thermo Xcalibur Workstation software (version 2.2.44). Data processing was performed with Thermo Scientific’s SIEVE 2.1, XCMS R Script [1921], and MetaboAnalyst 3.0 online [22,23]. Proteowizard’s MS Convert (version 3.0.5759) was used to centroid and convert Thermo (.raw) files into mzXML data formats for XCMS R processing. The background was subtracted from all acquired LC-MS raw data files using a random blank chromatogram from the same batch of analysis in the SIEVE program. Peaks that showed S/N ratios greater than 10 were selected as the detected compounds to be used in further analysis. P-values
